The present invention relates to DNA molecules encoding herbicide-tolerant forms of the enzyme protoporphyrinogen oxidase (xe2x80x9cprotoxxe2x80x9d). The invention further relates to herbicide-tolerant plants as well as methods for tissue culture selection and herbicide application based on these herbicide-tolerant forms of protox.
I. The Protox Enzyme and Its Involvement in the Chlorophyll/Heme Biosynthetic Pathway
The biosynthetic pathways that lead to the production of chlorophyll and heme share a number of common steps. Chlorophyll is a light harvesting pigment present in all green photosynthetic organisms. Heme is a cofactor of hemoglobin, cytochromes, P450 mixed-function oxygenases, peroxidases, and catalyses (see, e.g. Lehninger, Biochemistry, Worth Publishers, New York (1975)), and is therefore a necessary component for all aerobic organisms.
The last common step in chlorophyll and heme biosynthesis is the oxidation of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase (referred to, herein as xe2x80x9cprotoxxe2x80x9d) is the enzyme that catalyzes this last oxidation step (Matringe et al., Biochem J. 260: 231 (1989)).
The protox enzyme has been purified either partially or completely from a number of organisms including the yeast Saccharomyces cerevisiae (Labbe-Bois and Labbe, In Biosynthesis of Heme and Chlorophyll, E. H. Dailey, ed. McGraw Hill: New York, pp. 235-285 (1990)), barley etioplasts (Jacobs and Jacobs, Biochem J. 244: 219 (1987)), and mouse liver (Dailey and Karr, Biochem. 26: 2697 (1987)). Genes encoding protox have been isolated from two prokaryotic organisms, Escherichia coli (Sasarman et al., Can. J. Microbiol. 39: 1155 (1993)) and Bacillus subtilis (Dailey et al., J. Biol. Chem. 269: 813 (1994)). These genes share no sequence similarity; neither do their predicted protein products share any amino acid sequence identity. The E. coli protein is approximately 21 kDa, and associates with the cell membrane. The B. subtilis protein is 51 kDa, and is a soluble, cytoplasmic activity.
Protox encoding genes have now also been isolated from humans (see Nishimura et al., J. Biol. Chem. 270(14): 8076-8080 (1995) and plants (International application no. PCT/IB95/00452 filed Jun. 8, 1995, published Dec. 21, 1995 as WO 95/34659).
II. The Protox Gene As a Herbicide Target
The use of herbicides to control undesirable vegetation such as weeds or plants in crops has become an almost universal practice. The relevant market exceeds a billion dollars annually. Despite this extensive use, weed control remains a significant and costly problem for farmers.
Effective use of herbicides requires sound management. For instance, time and method of application and stage of weed plant development are critical to getting good weed control with herbicides. Since various weed species are resistant to herbicides, the production of effective herbicides becomes increasingly important. Novel herbicides can now be discovered using high-throughput screens that implement recombinant DNA technology. Metabolic enzymes essential to plant growth and development can be recombinantly produced though standard molecular biological techniques and utilized as herbicide targets in screens for novel inhibitors of the enzymes"" activity. The novel inhibitors discovered through such screens may then be used as herbicides to control undesirable vegetation.
Unfortunately, herbicides that exhibit greater potency, broader weed spectrum and more rapid degradation in soil can also have greater crop phytotoxicity. One solution applied to this problem has been to develop crops that are resistant or tolerant to herbicides. Crop hybrids or varieties resistant to the herbicides allow for the use of the herbicides without attendant risk of damage to the crop. Development of resistance can allow application of a herbicide to a crop where its use was previously precluded or limited (e.g. to pre-emergence use) due to sensitivity of the crop to the herbicide. For example, U.S. Pat. No. 4,761,373, incorporated herein by reference, is directed to plants resistant to various imidazolinone or sulfonamide herbicides. The resistance is conferred by an altered acetohydroxyacid synthase (AHAS) enzyme. U.S. Pat. No. 4,975,374, incorporated herein by reference, relates to plant cells and plants containing a gene encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that were known to inhibit GS, e.g. phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,013,659, incorporated herein by reference, is directed to plants that express a mutant acetolactate synthase (ALS) that renders the plants resistant to inhibition by sulfonylurea herbicides. U.S. Pat. No. 5,162,602, incorporated herein by reference, discloses plants tolerant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The tolerance is conferred by an altered acetyl coenzyme A carboxylase(ACCase). U.S. Pat. No. 5,554,798, incorporated herein by reference, discloses transgenic glyphosate resistant maize plants, which tolerance is conferred by an altered 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene.
The protox enzyme serves as the target for a variety of herbicidal compounds. The herbicides that inhibit protox include many different structural classes of molecules (Duke et al., Weed Sci. 39: 465 (1991); Nandihalli et al., Pesticide Biochem. Physiol. 43: 193 (1992); Matringe et al, FEBS Lett. 245: 35 (1989); Yanase and Andoh, Pesticide Biochem. Physiol. 35: 70 (1989)). These herbicidal compounds include the diphenylethers {e.g. acifluorfen, 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobezoic acid; its methyl ester, or oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles, (e.g. oxidiazon, 3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one), cyclic imides (e.g. S-23142,N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide), phenyl pyrazoles (e.g. TNPP-ethyl, ethyl 2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate; MandB 39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and its O-phenylpyrrolidino- and piperidinocarbamate analogs. Many of these compounds competitively inhibit the normal reaction catalyzed by the enzyme, apparently acting as substrate analogs.
Typically, the inhibitory effect on protox is determined by measuring fluorescence at about 622 to 635 nm, after excitation at about 395 to 410 nM (see, e.g. Jacobs and Jacobs, Enzyme 28: 206 (1982); Sherman et al., Plant Physiol. 97: 280 (1991)). This assay is based on the fact that protoporphyrin IX is a fluorescent pigment, and protoporphyrinogen IX is nonfluorescent.
The predicted mode of action of protox-inhibiting herbicides involves the accumulation of protoporphyrinogen IX in the chloroplast. This accumulation is thought to lead to leakage of protoporphyrinogen IX into the cytosol where it is oxidized by a peroxidase activity to protoporphyrin IX. When exposed to light, protoporphyrin IC can cause formation of singlet oxygen in the cytosol. This singlet oxygen can in turn lead to the formation of other reactive oxygen species, which can cause lipid peroxidation and membrane disruption leading to rapid cell death (Lee et al., Plant Physiol. 102: 881 (1993)).
Not all protox enzymes are sensitive to herbicides that inhibit plant protox enzymes. Both of the protox enzymes encoded by genes isolated from Escherchia coli (Sasarman et al., Can. J. Microbiol. 39: 1155 (1993)) and Bacillus subtilis (Dailey et al., J. Biol. Chem. 269: 813 (1994)) are resistant to these herbicidal inhibitors. In addition, mutants of the unicellular alga Chlamydomonas reinhardtii resistant to the phenylimide herbicide S-23142 have been reported (Kataoka et al., J. Pesticide Sci. 15: 449 (1990); Shibata et al., In Research in Photosynthesis, Vol. III, N. Murata, ed. Kluwer:Netherlands. pp. 567-570 (1992)). At least one of these mutants appears to have an altered protox activity that is resistant not only to the herbicidal inhibitor on which the mutant was selected, but also to other classes of protox inhibitors (Oshio et al., Z. Naturforsch. 48c: 339 (1993); Sato et al., In ACS Symposium on Porphyric Pesticides, S. Duke, ed. ACS Press: Washington, D.C. (1994)). A mutant tobacco cell line has also been reported that is resistant to the inhibitor S-21432 (Che et al.,. Z. Naturforsch 48c: 350 (1993).
III. Plastid Transformation and Expression
Plastid transformation, in which genes are inserted by homologous recombination into some or all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that may exceed 10% of the total soluble plant protein. In addition, plastid transformation is desirable because in most plants plastid-encoded traits are not pollen transmissible; hence, potential risks of inadvertent transgene escape to wild relatives of transgenic plants is obviated. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,576,198; in PCT Application Nos. WO 95/16783 and WO 97/32977; and in McBride et al., Proc. Natl. Acad. Sci. USA 91: 7301-7305 (1994), all of which are incorporated herein by reference. Plastid transformation via biolistics was achieved initially in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al. (1988) Science 240: 1534-1537, incorporated herein by reference) and this approach, using selection for cis-acting antibiotic resistance loci (spectinomycin/streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes, was soon extended to Nicotiana tabacum (Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8526-8530, incorporated herein by reference).
The basic technique for tobacco chloroplast transformation involves the particle bombardment of leaf tissue or PEG-mediated uptake of plasmid DNA in protoplasts with regions of cloned plastid DNA flanking a selectable antibiotic resistance marker. The 1 to 1.5 kb flanking regions, termed xe2x80x9ctargeting sequences,xe2x80x9d facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome. Initially, point mutations in the chloroplast 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin were utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45, incorporated herein by reference). This resulted in stable homoplasrnic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P., EMBO J. 12: 601-606 (1993), incorporated herein by reference). Substantial increases in transformation frequency were obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3xe2x80x2-adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917, incorporated herein by reference). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19, 4083-4089, incorporated herein by reference). Recently, plastid transformation of protoplasts from tobacco and the moss Physcomitrella patens has been attained using polyethylene glycol (PEG) mediated DNA uptake (O""Neill et al. (1993) Plant J. 3: 729-738; Koop et al. (1996) Planta 199: 193-201, both of which are incorporated herein by reference).
The present invention provides DNA molecules isolated from wheat, soybean, cotton, ugar beet, oilseed rape, rice, sorghum, and sugar cane encoding enzymes having protoporphyrinogen oxidase (protox) activity and chimeric genes comprising such DNA. Sequences of such DNA molecules are set forth in SEQ ID NOs: 9 (wheat), 11 (soybean), 15 (cotton), 17 (sugar beet), 19 (oilseed rape), 21 (rice), 23 (sorghum), and 36 (sugar cane).
The present invention also provides modified forms of plant protoporphyrinogen oxidase (protox) enzymes that are resistant to compounds that inhibit unmodified naturally occurring plant protox enzymes, and DNA molecules coding for such inhibitor-resistant plant protox enzymes. Thus, in one aspect the present invention provides a DNA molecule encoding a plant protox enzyme that is capable of being incorporated into a DNA construct used to transform a plant containing wild-type, herbicide-sensitive protox, wherein the DNA molecule has at least one point mutation relative to a wild-type DNA molecule encoding plant protox such that upon transformation with the DNA construct the plant contains the DNA molecule, which renders the plant resistant to the application of a herbicide that inhibits naturally occurring plant protox. The present invention includes chimeric genes and modified forms of naturally occurring protox genes that can express the inhibitor-resistant plant protox enzymes in plants.
Genes encoding inhibitor-resistant plant protox enzymes can be used to confer resistance to protox-inhibitory herbicides in whole plants and as a selectable marker in plant cell transformation methods. Accordingly, the present invention also includes plants, including the descendants thereof, plant tissues and plant seeds containing plant expressible genes encoding these modified protox enzymes. These plants, plant tissues and plant seeds are resistant to protox-inhibitors at levels that normally are inhibitory to the naturally occurring protox activity in the plant. Plants encompassed by the invention especially include those that would be potential targets for protox inhibiting herbicides, particularly agronomically important crops such as maize and other cereal crops such as barley, wheat, sorghum, rye, oats, turf and forage grasses, millet and rice. Also comprised are other crop plants such as sugar cane, soybean, cotton, sugar beet, oilseed rape and tobacco.
The present invention accordingly provides a method for selecting plant cells transformed with a DNA molecule of the invention that encodes a herbicide-tolerant form of plant protox. The method comprises introducing the DNA molecule into plant cells whose growth is sensitive to inhibition by herbicides to which the protox encoded by the DNA molecule is resistant, thus forming a transformed plant cell. The transformed plant cell whose growth is resistant to the selected herbicide is identified by selection at a herbicide concentration that inhibits the growth of untransformed plant cells.
The present invention is directed further to methods for the production of plants, including plant material, such as for example plant tissues, protoplasts, cells, calli, organs, plant seeds, embryos, pollen, egg cells, zygotes, together with any other propagating material and plant parts, such as for example flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed by means of the process of the invention, which produce an inhibitor-resistant form of the plant protox enzyme provided herein. Such plants may be stably transformed with a structural gene encoding the resistant protox, or prepared by direct selection techniques whereby herbicide resistant lines are isolated, characterized and developed.
In another aspect, the present invention is directed to a method for controlling unwanted vegetation growing at a locus where a herbicide-tolerant, agronomically useful plant, which is transformed with a DNA molecule according to the present invention that encodes a herbicide-tolerant form of plant protox, has been cultivated. The method comprises applying to the locus to be protected an effective amount of herbicide that inhibits naturally occurring protox activity.
The present invention is further directed to probes and methods for detecting the presence of genes encoding inhibitor-resistant forms of the plant protox enzyme and quantitating levels of inhibitor-resistant protox transcripts in plant tissue. These methods may be used to identify or screen for plants or plant tissue containing and/or expressing a gene encoding an inhibitor-resistant form of the plant protox enzyme.
The present invention also relates to plastid transformation and to the expression of DNA molecules in a plant plastid. In a preferred embodiment, a native plant protox enzyme or a modified plant protox enzyme is expressed in plant plastids to obtain herbicide resistant plants.
In a further embodiment, the present invention is directed to a chimeric gene comprising: (a) a DNA molecule isolated from a plant, which in its native state encodes a polypeptide that comprises a plastid transit peptide, and a mature enzyme that is natively targeted to a plastid of the plant by the plastid transit peptide, wherein the DNA molecule is modified such that it does not encode a functional plastid transit peptide; and (b) a promoter capable of expressing the DNA molecule in a plastid, wherein the promoter is operatively linked to the DNA molecule. The DNA molecule may be modified in that at least a portion of the native plastid transit peptide coding sequence is absent from the DNA molecule. Alternatively, the DNA molecule may be modified in that one or more nucleotides of the native plastid transit peptide coding sequence are mutated, thereby rendering an encoded plastid transit peptide nonfunctional. The present invention also relates to plants homoplasmic for chloroplast genomes containing such chimeric genes. In a preferred embodiment, the DNA molecule encodes an enzyme that is naturally inhibited by a herbicidal compound. In this case, such plants are resistant to a herbicide that naturally inhibits the enzyme encoded by a DNA molecule according to the present invention.
The present invention is also directed to plants made resistant to a herbicide by transforming their plastid genome with a DNA molecule according to the present invention and to methods for obtaining such plants. In a preferred embodiment, the DNA molecule encodes an enzyme that is naturally inhibited by a herbicidal compound. In a more preferred embodiment, the DNA molecule encodes an enzyme having protoporphyrinogen oxidase (protox) activity, which is modified so that it that confers resistance to protox inhibitors. A further embodiment of the present invention is directed to a method for controlling the growth of undesired vegetation, which comprises applying to a population of the above-described plants an effective amount of an inhibitor of the enzyme.
The present invention also provides a novel method for selecting a transplastomic plant cell, comprising the steps of: introducing the above-described chimeric gene into the plastome of a plant cell; expressing the encoded enzyme in the plastids of the plant cell; and selecting a cell that is resistant to a herbicidal compound that naturally inhibits the activity of the enzyme, whereby the resistant cell comprises transformed plastids. In a preferred embodiment, the enzyme is naturally inhibited by a herbicidal compound and the transgenic plant is able to grow on an amount of the herbicidal compound that naturally inhibits the activity of the enzyme. In a further preferred embodiment, the enzyme has protoporphyrinogen oxidase (protox) activity and is modified so that it that confers resistance to protox inhibitors.
SEQ ID NO:1: DNA coding sequence for an Arabidopsis thaliana protox-1 protein.
SEQ ID NO:2: Arabidopsis protox-1 amino acid sequence encoded by SEQ ID NO:1.
SEQ ID NO:3: DNA coding sequence for an Arabidopsis thaliana protox-2 protein.
SEQ ID NO:4: Arabidopsis protox-2 amino acid sequence encoded by SEQ ID NO:3.
SEQ ID NO:5: DNA coding sequence for a maize protox-1 protein.
SEQ ID NO:6: Maize protox-1 amino acid sequence encoded by SEQ ID NO:5.
SEQ ID NO:7: DNA coding sequence for a maize protox-2 protein.
SEQ ID NO:8: Maize protox-2 amino acid sequence encoded by SEQ ID NO:7.
SEQ ID NO:9: Partial DNA coding sequence for a wheat protox-1 protein.
SEQ ID NO:10: Partial wheat protox-1 amino acid sequence encoded by SEQ ID NO:9.
SEQ ID NO:11: DNA coding sequence for a soybean protox-1 protein.
SEQ ID NO:12: Soybean protox-1 protein encoded by SEQ ID NO:11.
SEQ ID NO:13: Promoter sequence from Arabidopsis thaliana protox-1 gene.
SEQ ID NO:14: Promoter sequence from maize protox-1 gene.
SEQ ID NO:15: DNA coding sequence for a cotton protox-1 protein.
SEQ ID NO:16: Cotton protox-1 amino acid sequence encoded by SEQ ID NO: 15.
SEQ ID NO:17: DNA coding sequence for a sugar beet protox-1 protein.
SEQ ID NO:18: Sugar beet protox-1 amino acid sequence encoded by SEQ ID NO: 17.
SEQ ID NO:19: DNA coding sequence for an oilseed rape protox-1 protein.
SEQ ID NO:20: Oilseed rape protox-1 amino acid sequence encoded by SEQ ID NO:19.
SEQ ID NO:21: Partial DNA coding sequence for a rice protox-1 protein.
SEQ ID NO:22: Partial rice protox-1 amino acid sequence encoded by SEQ ID NO:21.
SEQ ID NO:23: Partial DNA coding sequence for a sorghum protox-1 protein.
SEQ ID NO:24: Partial sorghum protox-1 amino acid sequence encoded by SEQ ID NO:23.
SEQ ID NO:25: Maize protox-1 intron sequence.
SEQ ID NO:26: Promoter sequence from sugar beet protox-1 gene.
SEQ ID NO:27: Pclp_P1axe2x80x94plastid clpP gene promoter top strand PCR primer.
SEQ ID NO:28: Pclp_P1bxe2x80x94plastid clpP gene promoter bottom strand PCR primer.
SEQ ID NO:29: Pclp_P2bxe2x80x94plastid clpP gene promoter bottom strand PCR primer.
SEQ ID NO:30: Trps16_P1axe2x80x94plastid rps16 gene top strand PCR primer.
SEQ ID NO:31: Trps16_p1bxe2x80x94plastid rps16 gene bottom strand PCR primer.
SEQ ID NO:32: minpsb_Uxe2x80x94plastid psbA gene top strand primer.
SEQ ID NO:33: minpsb_Lxe2x80x94plastid psbA gene bottom strand primer.
SEQ ID NO:34: APRTXP1axe2x80x94top strand PCR primer.
SEQ ID NO:35: APRIXP1bxe2x80x94bottom strand PCR primer.
SEQ ID NO:36: Partial DNA coding sequence for a sugar cane protox-1 protein.
SEQ ID NO:37: Partial sugar cane protox-1 amino acid sequence encoded by SEQ ID NO:36.
The following vector molecules have been deposited with Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, U.S.A on the dates indicated below:
Wheat protox-1a, in the pBluescript SK vector, was deposited Mar. 19, 1996, as pWDC-13 (NRRL #B21545).
Soybean protox-1, in the pBluescript SK vector, was deposited Dec. 15, 1995 as pWDC-12 (NRRL #B-21516).
Cotton protox-1, in the pBluescript SK vector, was deposited Jul. 1, 1996 as pWDC-15 (NRRL #B-21594).
Sugar beet protox-1, in the pBluescript SK vector, was deposited Jul. 29, 1996, as pWDC-16 (NRRL #B-21595N).
Oilseed rape protox-1, in the pBluescript SK vector, was deposited Aug. 23, 1996, as pWDC-17 (NRRL #B-21615).
Rice protox-1, in the pBluescript SK vector, was deposited Dec. 6, 1996, as pWDC-18 (NRRL #B-21648).
Sorghum protox-1, in the pBluescript SK vector, was deposited Dec. 6, 1996, as pWDC-19 (NRRL #B-21649).
Resistant mutant pAraC-2Cys, in the pMut-1 plasmid, was deposited on Nov. 14, 1994 under the designation pWDC-7 with the Agricultural Research Culture Collection and given the deposit designation NRRL #21339N.
AraPT1Pro containing the Arabidopsis protox-1 promoter was deposited Dec. 15, 1995, as pWDC-1 (NRRL #B-21515)
A plasmid containing the maize protox-1 promoter fused to the remainder of the maize protox-1 coding sequence was deposited Mar. 19, 1996 as pWDC-14 (NRRL #B-21546).
A plasmid containing the sugar beet protox-1 promoter was deposited Dec. 6, 1996, as pWDC-20 (NRRL #B-21650).
For clarity, certain terms used in the specification are defined and presented as follows:
Associated With/Operatively Linked: refers to two DNA sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be xe2x80x9cassociated withxe2x80x9d a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
Chimeric Gene: a recombinant DNA sequence in which a promoter or regulatory DNA sequence is operatively linked to, or associated with, a DNA sequence that codes for an mRNA or which is expressed as a protein, such that the regulator DNA sequence is able to regulate transcription or expression of the associated DNA sequence. The regulator DNA sequence of the chimeric gene is not normally operatively linked to the associated DNA sequence as found in nature.
Coding DNA Sequence: a DNA sequence that is translated in an organism to produce a protein.
Corresponding To: in the context of the present invention, xe2x80x9ccorresponding toxe2x80x9d means that when the amino acid sequences of various protox enzymes are aligned with each other, such as in Table 1A, the amino acids that xe2x80x9ccorrespond toxe2x80x9d certain enumerated positions in Table 1A are those that align with these positions in Table 1A, but that are not necessarily in these exact numerical positions relative to the particular protox enzyme""s amino acid sequence. Likewise, when the amino acid sequence of a particular protox enzyme (for example, the soybean protox enzyme) is aligned with the amino acid sequence of a reference protox enzyme (for example, the Arabidopsis protox-1 sequence given in SEQ ID NO:2), the amino acids in the soybean protox sequence that xe2x80x9ccorrespond toxe2x80x9d certain enumerated positions of SEQ ID NO:2 are those that align with these positions of SEQ ID NO:2, but are not necessarily in these exact numerical positions of the soybean protox enzyme""s amino acid sequence.
DNA Shuffling: DNA shuffling is a method to introduce mutations or rearrangements, preferably randomly, in a DNA molecule or a method to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a xe2x80x9cshuffled DNA molecule,xe2x80x9d that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule. The shuffled DNA encodes an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.
Herbicide: a chemical substance used to kill or suppress the growth of plants, plant cells, plant seeds, or plant tissues.
Heterologous DNA Sequence: a DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.
Homologous DNA Sequence: a DNA sequence naturally associated with a host cell into which it is introduced.
Homoplasmic: refers to a plant, plant tissue or plant cell, wherein all of the plastids are genetically identical. In different tissues or stages of development, the plastids may take different forms, e.g., chloroplasts, proplastids, etioplasts, amyloplasts, chromoplasts, and so forth.
Inhibitor: a chemical substance that inactivates the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the plant. In the context of the instant invention, an inhibitor is a chemical substance that inactivates the enzymatic activity of protox. The term xe2x80x9cherbicidexe2x80x9d is used herein to define an inhibitor when applied to plants, plant cells, plant seeds, or plant tissues.
Isolated: in the context of the present invention, an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
Minimal Promoter: promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcrption factor, the minimal promoter functions to permit transcription.
Modified Enzyme Activity: enzyme activity different from that which naturally occurs in a plant (i.e. enzyme activity that occurs naturally in the absence of direct or indirect manipulation of such activity by man), which is tolerant to inhibitors that inhibit the naturally occurring enzyme activity.
Nucleic Acid Molecule: a linear segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.
Plant: refers to any plant or part of a plant at any stage of development. Therein are also included cuttings, cell or tissue cultures and seeds. As used in conjunction with the present invention, the term xe2x80x9cplant tissuexe2x80x9d includes, but is not limited to, whole plants, plant cells, plant organs, plant seeds, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and/or functional units.
Plastome: the genome of a plastid.
Protox-1: chloroplast protox.
Protox-2: mitochondrial protox.
Significant Increase: an increase in enzymatic activity that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater of the activity of the wild-type enzyme in the presence of the inhibitor, more preferably an increase by about 5-fold or greater, and most preferably an increase by about 10-fold or greater.
Substantially Similar: with respect to nucleic acids, a nucleic acid molecule that has at least 60 percent sequence identity with a reference nucleic acid molecule. In a preferred embodiment, a substantially similar DNA sequence is at least 80% identical to a reference DNA sequence; in a more preferred embodiment, a substantially similar DNA sequence is at least 90% identical to a reference DNA sequence; and in a most preferred embodiment, a substantially similar DNA sequence is at least 95% identical to a reference DNA sequence. A substantially similar nucleotide sequence typically hybridizes to a reference nucleic acid molecule, or fragments thereof, under the following conditions: hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 pH 7.0, 1 mM EDTA at 50xc2x0 C.; wash with 2xc3x97SSC, 1% SDS, at 50xc2x0 C. With respect to proteins or peptides, a substantially similar amino acid sequence is an amino acid sequence that is at least 90% identical to the amino acid sequence of a reference protein or peptide and has substantially the same activity as the reference protein or peptide.
Tolerance/Resistance: the ability to continue normal growth or function when exposed to an inhibitor or herbicide.
Transformation: a process for introducing heterologous DNA into a cell, tissue, or plant. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
Transit Peptide: a signal polypeptide that is translated in conjunction with a protein encoded by a DNA molecule, forming a polypeptide precursor. In the process of transport to a selected site within the cell, a chloroplast for example, the transit peptide can be cleaved from the remainder of the polypeptide precursor to provide an active or mature protein.
Transformed: refers to an organism such as a plant into which a heterologous DNA molecule has been introduced. The DNA molecule can be stably integrated into the genome of the plant, wherein the genome of the plant encompasses the nuclear genome, the plastid genome and the mitochondrial genome. In a transformed plant, the DNA molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. A xe2x80x9cnon-transformedxe2x80x9d plant refers to a wild-type organism, i.e., a plant, which does not contain the heterologous DNA molecule.
Transplastome: a transformed plastid genome.
Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (M), and guanine (G). Amino acids are likewise indicated by the following standard abbreviations: alanine (ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser. S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Furthermore (Xaa; X) represents any amino acid.